Simultaneous quantification of hepatitis A virus and norovirus genogroup I and II by triplex droplet digital PCR

Han, Yanzhen; Wang, Jianchang; Zhang, Shuhong; Yang, Shuopeng; Wang, Xiangji; Han, Yanqing; Shen, Zhixin; Xu, Xiangdong

doi:10.1016/j.fm.2021.103933

Cited by 8 publications

(6 citation statements)

References 40 publications

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“…We must note that the reliability of the quantification was ensured by the linearity of all the curves, with R2 values always >0.95. These values are below those reported by other authors, such as Han et al [ 21 ], who reported a LOQ of 20 or 25 cps/rctn, depending on the virus (hepatitis A or norovirus g.I or g.II), or Mairiang et al [ 22 ], who obtained a LOQ of 2.337, but in terms of Loq 10 cps/rctn, actually corresponds to 217 cps/rctn.…”

Section: Discussioncontrasting

confidence: 71%

Designing and Validation of a Droplet Digital PCR Procedure for Diagnosis and Accurate Quantification of Nervous Necrosis Virus in the Mediterranean Area

Souto,

Olveira,

López-Vázquez

et al. 2023

Pathogens

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The viral nervous necrosis virus (VNNV) is the causative agent of an important disease affecting fish species cultured worldwide. Early and accurate diagnosis is, at present, the most effective control and prevention tool, and molecular techniques have been strongly introduced and accepted by official organizations. Among those, real-time quantitative polymerase chain reaction (rt-qPCR) is nowadays displacing other molecular techniques. However, another PCR-based technology, droplet digital PCR (ddPCR), is on the increase. It has many advantages over qPCR, such as higher sensitivity and more reliability of the quantification. Therefore, we decided to design and validate a protocol for the diagnosis and quantification of SJ and RG type VNNV using reverse transcription-ddPCR (RT-ddPCR). We obtained an extremely low limit of detection, 10- to 100-fold lower than with RT-qPCR. Quantification by RT-ddPCR, with a dynamic range of 6.8–6.8 × 104 (SJ type) or 1.04 × 101–1.04 × 105 (RG type) cps/rctn, was more reliable than with RT-qPCR. The procedure was tested and validated in field samples, providing high clinical sensitivity and negative predictive values. In conclusion, we propose this method to substitute RT-qPCR protocols because it exceeds the expectations of qPCR in the diagnosis and quantification of VNNV.

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Section: Discussioncontrasting

confidence: 71%

Designing and Validation of a Droplet Digital PCR Procedure for Diagnosis and Accurate Quantification of Nervous Necrosis Virus in the Mediterranean Area

Souto,

Olveira,

López-Vázquez

et al. 2023

Pathogens

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“…In this study, the sensitivity of RT-ddPCR method was lower than in our previous work detecting for Norovirus and Hepatitis A Virus [ 28 ], likely due to the one-step method we used in this study. Previously, Racˇki et al found that the degree of inhibition of one-step RT-ddPCR amplification of RNA by undiluted raw wastewater is the same as that of RT-qPCR determination [ 29 ].…”

Section: Discussionmentioning

confidence: 55%

Surveillance of SARS-CoV-2 in wastewater by quantitative PCR and digital PCR: a case study in Shijiazhuang city, Hebei province, China

Chai,

Liu,

Liu

et al. 2024

Emerging Microbes & Infections

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In this study, we reported the first long-term monitoring of SARS-CoV-2 in wastewater in Mainland China from November 2021 to October 2023. The city of Shijiazhuang was employed for this case study. We developed a triple reverse transcription droplet digital PCR (RT-ddPCR) method using triple primer-probes for simultaneous detection of the N1 gene, E gene, and Pepper mild mottle virus (PMMoV) to achieve accurate quantification of SARS-CoV-2 RNA in wastewater. Both the RT-ddPCR method and the commercial multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) method were implemented for the detection of SARS-CoV-2 in wastewater in Shijiazhuang City over a 24-month period. Results showed that SARS-CoV-2 was detected for the first time in the wastewater of Shijiazhuang City on 10 November 2022. The peak of COVID-19 cases occurred in the middle of December 2022, when the concentration of SARS-CoV-2 in the wastewater was highest. The trend of virus concentration increases and decreases forming a “long-tailed” shape in the COVID-19 outbreak and recession cycle. The results indicated that both multiplex RT-ddPCR and RT-qPCR are effective in detecting SARS-CoV-2 in wastewater, but RT-ddPCR is capable of detecting low concentrations of SARS-CoV-2 in wastewater which is more efficient. The SARS-CoV-2 abundance in wastewater is correlated to clinical data, outlining the public health utility of this work. Highlights First long-term monitoring of SARS-CoV-2 in wastewater in Mainland China COVID-19 outbreak was tracked in Shijiazhuang City from outbreak to containment Wastewater was monitored simultaneously using RT-ddPCR and RT-qPCR methods Triple primer-probe RT-ddPCR detects N1 and E genes of SARS-CoV-2 and PMMoV

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“…Similar to qPCR, dPCR allows the simultaneous quantification of different targets. HAV and norovirus genogroup I and II were quantified simultaneously by triplex droplet reverse transcription digital PCR in food, drinking water, and faecal samples [ 210 ]. In general, RT-dPCR can be more expensive and time consuming compared to RT-qPCR, but no external calibration curves are needed for the quantification.…”

Section: Methods Used For Detection Of Hav and Hev In Different Food ...mentioning

confidence: 99%

Hepatitis A Virus and Hepatitis E Virus as Food- and Waterborne Pathogens—Transmission Routes and Methods for Detection in Food

Nemes

Persson

Simonsson

2023

Viruses

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Foodborne viruses are an important threat to food safety and public health. Globally, there are approximately 5 million cases of acute viral hepatitis due to hepatitis A virus (HAV) and hepatitis E virus (HEV) every year. HAV is responsible for numerous food-related viral outbreaks worldwide, while HEV is an emerging pathogen with a global health burden. The reported HEV cases in Europe have increased tenfold in the last 20 years due to its zoonotic transmission through the consumption of infected meat or meat products. HEV is considered the most common cause of acute viral hepatitis worldwide currently. This review focuses on the latest findings on the foodborne transmission routes of HAV and HEV and the methods for their detection in different food matrices.

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Simultaneous quantification of hepatitis A virus and norovirus genogroup I and II by triplex droplet digital PCR

Cited by 8 publications

References 40 publications

Designing and Validation of a Droplet Digital PCR Procedure for Diagnosis and Accurate Quantification of Nervous Necrosis Virus in the Mediterranean Area

Designing and Validation of a Droplet Digital PCR Procedure for Diagnosis and Accurate Quantification of Nervous Necrosis Virus in the Mediterranean Area

Surveillance of SARS-CoV-2 in wastewater by quantitative PCR and digital PCR: a case study in Shijiazhuang city, Hebei province, China

Hepatitis A Virus and Hepatitis E Virus as Food- and Waterborne Pathogens—Transmission Routes and Methods for Detection in Food

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